The ability to produce a polypeptide or virus of interest is increasingly important to the biotechnology industry. Over the last two decades, advances in biotechnology have led to interest in numerous polypeptides and viruses that have potential therapeutic uses as vaccines and pharmaceuticals. Large scale production generally has involved recombinant production of the polypeptide or virus of interest, e.g., in bacterial, yeast, insect, mammalian, or other cell types. The production of polypeptides or viruses of interest in mammalian cultures, in particular, has advantages over production in bacterial or other lower microbial hosts because of the ability of mammalian cells to post-translationally process complex protein structures, via, e.g, disulfide-dependent folding and glycosylation.
Mammalian cells are generally propagated in vitro in one of two modes: as non-anchorage dependent cells growing freely in suspension throughout the bulk of the culture; or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth). Microcarrier systems have been developed to accommodate both types of growth. For example, anchorage-dependent cells may be propagated in microcarrier systems comprising small solid particles suspended in growth medium by slow agitation. This system allows anchorage-dependent cells to attach to the surfaces of the suspended particles, and grow to confluency, while the microcarriers remain suspended in the growth medium. Alternatively, macroporous microcarriers may be used to contain non-anchorage dependent cells in bioreactors, e.g., by nonspecific attachment for the cells to the surface of such microcarriers. Microcarrier suspension cultures of either non-anchorage dependent cells or anchorage-dependent cells are the most widely used means of large scale production of cells and cell products.
Large-scale suspension cultures may be operated in a closed system, for example, as batch or fed-batch closed systems, which are more straightforward to operate and scale up than open systems. Typically, in a closed system, no cells, products, and/or waste products are removed (although air (e.g., oxygen) may be added and CO2 removed by aeration). The typical growth profile seen with batch growth systems involves a lag phase, followed by an exponential phase, a stationary phase, and a decline phase. In such batch systems, the environment is continuously changing, as nutrients are depleted and metabolites accumulate. In a fed batch system, key nutrients are continuously fed into the system to prolong the growth cycle although cells, products, by products, and waste products, including toxic metabolites, are not removed. Accordingly, production of a polypeptide or virus of interest by the batch or fed batch systems is limited by the accumulation of cells and harmful substances, such as toxic metabolites.
Large-scale suspension cultures may also be operated in an open system, e.g., a perfusion system or a chemostat system. In a perfusion system, fresh medium is perfused through the culture while the cells are retained with a variety of cell retention devices. Types of cell retention devices include, for example, microcarriers, fine mesh spin filters, hollow fibers, flat plate membrane filters, settling tubes, ultrasonic cell retention devices, and the like. Typically, perfusion cultures are designed to increase cell densities to a maximum, with cell retention devices designed and operated to have a cell retention rate of >90%. Such cultures typically reach cell densities of >2×107, which may have to be supplied with a cell culture medium feed at a dilution rate greater than about 2.0 d−1. However, the steady state of the system is hard to maintain because of the uncontrolled increase in biomass, and consistent production conditions are difficult to control and/or achieve.
Chemostat systems are operated with a continuous inflow of medium and an outflow of cells and products. In the chemostat system, there is no cell retention device, such that the concentration of cells in the bioreactor and the concentration of the cells in supernatant harvested from the bioreactor are substantially identical. Typically, culture medium is fed to the reactor at a predetermined and constant rate, maintaining a low dilution rate of the culture (typically 0.3 d−1 to 0.8 d−1). To prevent washout of cells, the dilution rate generally is chosen to be less than, and sometimes equal to, the maximum specific growth rate of the cells. Culture fluids containing cells, cell products, byproducts, waste products, etc., are removed at the same rate, or substantially the same rate. Chemostat systems typically provide for a high degree of control, since the cultures may equilibrate, i.e., reach a steady state at a specific growth rate equivalent to the dilution rate. This equilibration is determinative of the concentration of the cells, metabolites, waste products, expressed products (e.g. secreted proteins), etc. Specific growth rates in chemostat systems are typically lower than the maximum growth rate due to at least one limiting substrate. In some systems, however, steady states may be maintained at the maximum specific growth rates by controlling and adjusting biomass, e.g., in turbidstat systems of chemostat cultures. Preferably, such chemostat cultures contain a homogeneous distribution of cells (e.g., single cell suspensions) throughout the bioreactor. Compared to the perfusion system, however, the chemostat system typically results in lower cell densities. Furthermore, an inherent disadvantage of chemostat systems is that the feed of the cells can not be controlled independent of the cell densities in the bioreactor system.
Suspension cell cultures for producing recombinant proteins in serum-free and/or chemically-defined media are also limited in that the serum-free and/or chemically-defined media typically support slower growth rates compared to cells grown in media containing serum. Lowered growth rates in the culture means a lowered production of the polypeptide or virus of interest.
Accordingly, there remains a need for the development of cell culture systems capable of sustaining production of a polypeptide or virus of interest, especially for cultures that can be sustained for prolonged periods of time, for example, to meet demands for increased production at low costs. The present invention provides methods and compositions directed at meeting this and other needs.